evaluate the barcode efficiency to discriminate
duckweed species for seven pairs of
chloroplast-derived markers. It has shown that
the best molecular marker is the atpF-atpH
noncoding spacer that could correct identify 14
out of 19 species (Wang et al. 2010). Still, the
maker could not reach the complete discriminations, especially for closely related species even
with the combination of several markers. The
whole chloroplast genome as a barcode marker
has the power to provide higher resolution and
more polymorphism to identify species. The use
of whole chloroplast genomes as a barcode system was demonstrated in certain species (Douglas 1998), breaking through the previous
limitations.
Genetic information in plants stores in three
parts of nucleus, mitochondrion, and chloroplast
(plastid). Nuclear genome is a hub coordinating
the activities of both mitochondrion and chloroplast in spite of the cross-talk between them. The
mitochondrion is a membrane-bound organelle
that converts large molecules of carbohydrates,
proteins, or lipids into the energy of adenosine
triphosphate (ATP) by respiration. The chloroplast is a producer that allows plant to capture the
sun energy into energy-rich molecules. Its genome is a circular, double-stranded DNA molecule with thousands of copies in a cell
(Fig. 10.1). There are roughly 100 mitochondrial, 1000 chloroplast, and 2 nuclear genome
copies in a duckweed cell (Wang et al. 2012). It
is believed to have evolved from endosymbiosis
of a cyanobacterium, together with a massive
gene transfer from the chloroplast to the nucleus
(Timmis et al. 2004). The chloroplast genome is
responsible for encoding the key protein complexes involved in photosynthesis and other
metabolic processes. The genome sizes are relatively constant with a range of 107–218 Kb
(Daniell et al. 2016). The chloroplast genome is a
circular molecule containing a large sing copy, a
small single copy and two inverted repeats. The
gene content and gene structure are also highly
conserved, indicating their essential functions
throughout the plant evolution (Daniell et al.
2016).
The advent of high-throughput sequencing
technologies and bioinformatics tools has facilitated the rapid progress in the fields of chloroplast genetics and genomics. Until 2018, the
database of NCBI Organelle Genomes section
has collected 1975 annotated chloroplast/plastid
genomes from land plants (https://www.ncbi.
nlm.nih.gov/genome/organelle/). The first duckweed chloroplast genome of Lemna minor was
released in 2008 by using traditional Sanger
sequencing (Mardanov et al. 2008), and the other
three duckweed chloroplast genomes were
sequenced by next-generation sequencing
(NGS) in 2011 (Wang and Messing 2011). All
the sequences were deposited in NCBI database
and could be fetched with the unique ID number.
Here, we reviewed the complete chloroplast
genomes of four duckweed species, which gave
insights into the overall evolutionary dynamics
and phylogenetic relationship compared to other
plants. We also present the strategy of genome
assembly with the short reads from NGS and
prospected the long reads produced by
third-generation technology. The pipeline including DNA preparation, sequencing technology, and
computational tools for genome assembly and
annotation
were
informatively
covered
(Fig. 10.2). We expect the availability of more
duckweed chloroplast genomes would help our
understanding for the origins and features of
duckweed species, but shed new lights on their
evolution and biotechnological applications.
10.2 DNA Preparation
10.2.1 Pure cpDNA Isolation
10.2.1.1 Gradient Centrifuge
The high-quality and decent amount of starting
chloroplast DNA (cpDNA) is a prerequisite for
sequencing chloroplast genome, whereas cpDNA
isolation is tedious and time-consuming that has
restricted the broad applications. It is well known
that a plant cell contains three types of DNA
derived from nucleus, mitochondrion and
chloroplast. A regular CTAB way is to get the
104
Y. Zhang and W. Wang
duckweed species for seven pairs of
chloroplast-derived markers. It has shown that
the best molecular marker is the atpF-atpH
noncoding spacer that could correct identify 14
out of 19 species (Wang et al. 2010). Still, the
maker could not reach the complete discriminations, especially for closely related species even
with the combination of several markers. The
whole chloroplast genome as a barcode marker
has the power to provide higher resolution and
more polymorphism to identify species. The use
of whole chloroplast genomes as a barcode system was demonstrated in certain species (Douglas 1998), breaking through the previous
limitations.
Genetic information in plants stores in three
parts of nucleus, mitochondrion, and chloroplast
(plastid). Nuclear genome is a hub coordinating
the activities of both mitochondrion and chloroplast in spite of the cross-talk between them. The
mitochondrion is a membrane-bound organelle
that converts large molecules of carbohydrates,
proteins, or lipids into the energy of adenosine
triphosphate (ATP) by respiration. The chloroplast is a producer that allows plant to capture the
sun energy into energy-rich molecules. Its genome is a circular, double-stranded DNA molecule with thousands of copies in a cell
(Fig. 10.1). There are roughly 100 mitochondrial, 1000 chloroplast, and 2 nuclear genome
copies in a duckweed cell (Wang et al. 2012). It
is believed to have evolved from endosymbiosis
of a cyanobacterium, together with a massive
gene transfer from the chloroplast to the nucleus
(Timmis et al. 2004). The chloroplast genome is
responsible for encoding the key protein complexes involved in photosynthesis and other
metabolic processes. The genome sizes are relatively constant with a range of 107–218 Kb
(Daniell et al. 2016). The chloroplast genome is a
circular molecule containing a large sing copy, a
small single copy and two inverted repeats. The
gene content and gene structure are also highly
conserved, indicating their essential functions
throughout the plant evolution (Daniell et al.
2016).
The advent of high-throughput sequencing
technologies and bioinformatics tools has facilitated the rapid progress in the fields of chloroplast genetics and genomics. Until 2018, the
database of NCBI Organelle Genomes section
has collected 1975 annotated chloroplast/plastid
genomes from land plants (https://www.ncbi.
nlm.nih.gov/genome/organelle/). The first duckweed chloroplast genome of Lemna minor was
released in 2008 by using traditional Sanger
sequencing (Mardanov et al. 2008), and the other
three duckweed chloroplast genomes were
sequenced by next-generation sequencing
(NGS) in 2011 (Wang and Messing 2011). All
the sequences were deposited in NCBI database
and could be fetched with the unique ID number.
Here, we reviewed the complete chloroplast
genomes of four duckweed species, which gave
insights into the overall evolutionary dynamics
and phylogenetic relationship compared to other
plants. We also present the strategy of genome
assembly with the short reads from NGS and
prospected the long reads produced by
third-generation technology. The pipeline including DNA preparation, sequencing technology, and
computational tools for genome assembly and
annotation
were
informatively
covered
(Fig. 10.2). We expect the availability of more
duckweed chloroplast genomes would help our
understanding for the origins and features of
duckweed species, but shed new lights on their
evolution and biotechnological applications.
10.2 DNA Preparation
10.2.1 Pure cpDNA Isolation
10.2.1.1 Gradient Centrifuge
The high-quality and decent amount of starting
chloroplast DNA (cpDNA) is a prerequisite for
sequencing chloroplast genome, whereas cpDNA
isolation is tedious and time-consuming that has
restricted the broad applications. It is well known
that a plant cell contains three types of DNA
derived from nucleus, mitochondrion and
chloroplast. A regular CTAB way is to get the
104
Y. Zhang and W. Wang